Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-02-09
2001-09-25
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S375000, C436S063000, C436S064000
Reexamination Certificate
active
06294348
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns the isolation, recombinant production and characterization of novel polypeptides which block signal transduction mediated by tumor necrosis factor associated factors (TRAFs), including TRAF2. In particular, this invention concerns novel proteins that act as specific inhibitors of TRAF2-dependent NF-&kgr;B activation signaled by certain members of the TNF receptor superfamily, such as TNF-R2 and CD40, methods and means for making them and their use in screening assays or as pharmaceuticals.
BACKGROUND OF THE INVENTION
Tumor necrosis factor (TNF) is a cytokine produced mainly by activated macrophages which elicits a wide range of biological effects. These include an important role in endotoxic shock and in inflammatory, immunoregulatory, proliferative, cytotoxic, and anti-viral activities (reviewed by Goeddel, D. V. et al.,
Cold Spring Harbor Symposia on Quantitative Biology
51, 597-609 [1986]; Beutler, B. and Cerami, A.,
Ann. Rev. Biochem.
57, 505-518 [1988]; Old, L. J.,
Sci. Am.
258(5),59-75 [1988]; Fiers, W.
FEBS Lett.
285(2), 199-212 [1991]). The induction of the various cellular responses mediated by TNF is initiated by its interaction with two distinct cell surface receptors, an approximately 55 kDa receptor termed TNF-R1 and an approximately 75 kDa receptor termed TNF-R2. Human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher, H. et al.,
Cell
61, 351 [1990]; Schall, T. J. et al.,
Cell
61, 361 [1990]; Smith, C. A. et al.,
Science
248, 1019 [1990]; Lewis, M. et al.,
Proc. Natl. Acad. Sci. USA
88, 2830-2834 [1991]; Goodwin, R. G. et al.,
Mol. Cell. Biol.
11, 3020-3026 [1991]. Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions. The extracellular portions of both receptors are found naturally also as soluble TNF-binding proteins (Nophar, Y. et al.,
EMBO J.
9, 3269 [1990] and Kohno, T. et al.,
Proc. Natl. Acad. Sci. U.S.A.
87, 8331 [1990]). The amino acid sequence of human NF-R1 and the underlying nucleotide sequence are disclosed in EP 417,563 (published Mar. 20, 1991), whereas EP 418,014 (published Mar. 20, 1991) discloses the amino acid and nucleotide sequences of human TNF-R2.
Both TNF receptors are independently active in signaling TNF responses. Direct signaling by TNF-R2 has been observed in lymphoid cells in which TNF-R2 stimulates the proliferation of thymocytes and a murine cytotoxic T cell line CT6 (Tartaglia et al.,
Proc. Natl. Acad. Sci. USA
88, 9292-9296 [1991]; Tartaglia et al,
J. Immunol.
151, 4637-4641 [1993]). Both TNF-R1 and TNF-R2, along with other members of the TNF receptor superfamily, e.g. CD40, have been shown to independently mediate the activation of the transcription factor NF-&kgr;B (Lenardo & Baltimore,
Cell
58: 227-229 [1989]; Lægreid, A., et al.,
J. Biol. Chem.
269, 7785-7791 [1994]; Rothe et al.,
Cell
78, 681-692 [1994]; Wiegmann et al.,
J. Biol. Chem.
267, 17997-18001 [1992]). NF-&kgr;B is a member of the Re1 family of transcriptional activators that control the expression of a variety of important cellular and viral genes (Lenardo & Baltimore, supra, and Thanos and Maniatis,
Cell
80, 529-532 [1995]). TNF-R2 also mediates the transcriptional induction of the granulocyte-macrophage colony stimulating factor (GM-CSF) gene (Miyatake et al.,
EMBO J.
4: 2561-2568 [1985]; Stanley et al.,
EMBO J.
4: 2569-2573 [1985]) and the A20 zinc finger protein gene (Opipari et al.,
J. Biol. Chem.
265: 14705-14708 [1990]) in CT6 cells, and participates as an accessory component to TNF-R1 in the signaling of responses primarily mediated by TNF-R1, like cytotoxicity (Tartaglia, L. A. and Goeddel, D. V.,
Immunol. Today
13, 151-153 [1992]).
Recent research has lead to the isolation of polypeptide factors associated with the intracellular domain of the 75 kDa tumor necrosis factor receptor, TNF-R2 (“tumor necrosis factor receptor associated factors” or “TRAFs”) which participate in the TNF-R2 signal transduction cascade. TRAF1 and TRAF2 were the first two identified members of this novel protein family containing a novel C-terminal homology region, the TRAF domain (Rothe et al.,
Cell
78, 681-692 [1994]; U.S. Pat. Nos. 5,670,319 issued Sep. 23, 1997, and 5,708,142 issued Jan. 13, 1998. A further TRAF domain protein, TRAF3 (originally termed CD40 bp, CRAF, or LAP1) has also been identified (Hu et al.,
J. Biol. Chem.
269, 30069 [1994]; Cheng et al.,
Science
267, 1494 [1995], and Mosialos et al,
Cell
80, 389 [1995]). TRAFs transduce signals from TNF-R2, CD40 and presumably from other members of the TNF receptor superfamily that also includes the low affinity nerve growth factor receptor, the Fas antigen, CD27, CD30, OX40, 4-1BB, and TNFR-RP (Rothe et al., supra; Hu et al.,
J. Biol. Chem.
269, 30069-30072 [1994]; Cheng et al.,
Science
267, 1494-1498[1995]; Mosialos et al.,
Cell
80, 389-399 [1995]; Rothe et al.,
Science,
in press, Smith et al,
Cell
76, 959-962 [1994]). In addition to the shared conserved C-terminal TRAF domain that is involved in both receptor association and oligomerization, TRAF2 and TRAF3 each contain an N-terminal RING finger domain and five zinc finger structures of weak sequence similarity. CD40 and TNF-R2 interact directly with TRAF2 and indirectly with TRAF1 via a TRAF2/TRAF1 heterodimer. TRAF2 (or the TRAF2/TRAF1 heterodimer) is required for CD40- and TNF-R2-mediated activation of the transcription factor NF-&kgr;B. TRAF3 interacts with CD40 and self-associates,but does appear to not associate with TNF-R2, TRAF1, or TRAF2. The role of TRAF3 in signal transduction is less well defined, but it may antagonize the effects of TRAF2 (Rothe et al.,
Science,
in press). The TRAF proteins also interact with the C-terminal cytoplasmic domain of the Epstein-Barr virus transforming protein LMP1 (Mosialos et al.,
Cell
80, 389 [1995]). LMP1 is a dominant oncogene that has multiple downstream effects on cell growth and gene expression, at least some of which require NF-&kgr;B activation (Laherty et al.,
J. Biol. Chem.
267, 24157 [1992]; Rowe et al.,
J. Virol.
68, 5602 [1994]). TRAF2 is believed to be a common signal transducer for TNF-R2, CD40 and LMP1.
SUMMARY OF THE INVENTION
The present invention is based on the identification, recombinant production and characterization of certain novel TRAF-interacting proteins named “inhibitors of TRAF” (I-TRAFs). More specifically, the present invention is based on the isolation of cDNAs encoding various forms of murine and human I-TRAFs encoding proteins with no significant sequence similarity to any known protein, and on the expression and characterization of the encoded I-TRAF proteins.
In one aspect, the invention concerns purified I-TRAF proteins which inhibit the interaction of TRAFs with members of the TNF receptor superfamily, such as TNF-R2, CD40, and/or other cellular proteins with which TRAF proteins are normally, directly or indirectly, associated, such as LMP1.
In particular, the invention concerns an isolated I-TRAF polypeptide comprising the amino acid sequence of a polypeptide selected from the group consisting of:
(a) a native sequence I-TRAF polypeptide,
(b) a polypeptide having at least about 70% amino acid sequence identity with the TRAF-binding domain of a native sequence I-TRAF polypeptide and capable of inhibiting a TRAF-mediated signaling event, and
(c) a fragment of a polypeptide of (a) or (b) capable of inhibiting a TRAF-mediated signaling event.
Preferably, the I-TRAF polypeptides of the present invention have an at least about 60% overall amino acid sequence identity with a native sequence I-TRAF polypeptide. The I-TRAF p
Goeddel David V.
Rothe Mike
Dreger Ginger
Genentech Inc.
Marschang Diane L.
Scheiner Laurie
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